There is a compelling need to develop 3D assay systems for HTS formats to identify new potential anticancer agents. A key limitation is the difficulty in culturing and analyzing cells in 3D extracellular matrix (ECM) in the multiwell plates used for HTS assays. BellBrook Labs has developed an innovative alternative that relies on microchannels instead of wells to enable automated 3D cell culture and high content analysis in ECM. Our collaborators at the University of Wisconsin-Madison have developed an ECM microfabrication technology that has outstanding scientific and commercial synergy with the iuvo platform. They are able to custom fabricate ECM with Tumor Associated Collagen Signatures linked to metastatic potential using multiphoton methods that are compatible with iuvo plates. In Phase I we will 1) fabricate breast, ovarian, and pancreatic collagen-derived ECM in iuvo microchannel plates; 2) analyze cell behavior, including migration, invasion, and proliferation; 3) introduce tumor associated fibroblats and macrophages to the iuvoTACS platform; 4) benchmark cell responses compared against those in conventional 2D, and random 3D collagen gels; 5) determine concentration curves for response to anti-cancer agents with different ECM and stromal cell configurations. The combination of the UW team?s ECM technology with the iuvo plates is truly transformative, as it will allow scientists to probe tumor cell behavior in custom-fabricated ECM that faithfully replicates the in vivo tumor microenvironment.